Human adrenergic receptors are integral membrane proteins which have been classified into two broad classes, the alpha and the beta adrenergic receptors. Both types mediate the action of the peripheral sympathetic nervous system upon binding of catecholamines, norepinephrine and epinephrine.
Norepinephrine is produced by adrenergic nerve endings, while epinephrine is produced by the adrenal medulla. The binding affinity of adrenergic receptors for these compounds forms one basis of the classification: alpha receptors bind norepinephrine more strongly than epinephrine and much more strongly than the synthetic compound isoproterenol. The binding affinity of these hormones is reversed for the beta receptors. In many tissues, the functional responses, such as smooth muscle contraction, induced by alpha receptor activation are opposed to responses induced by beta receptor binding.
Subsequently, the functional distinction between alpha and beta receptors was further highlighted and refined by the pharmacological characterization of these receptors from various animal and tissue sources. As a result, alpha and beta adrenergic receptors were further subdivided into .alpha..sub.1, .alpha..sub.2, .beta..sub.1, and .beta..sub.2 subtypes. Functional differences between .alpha..sub.1 and .alpha..sub.2 receptors have been recognized, and compounds which exhibit selective binding between these two subtypes have been developed. Thus, in WO 92/0073, the selective ability of the R(+) enantiomer of terazosin to selectively bind to adrenergic receptors of the alpha 1 subtype was reported. The .alpha..sub.1 /.alpha..sub.2 selectivity of this compound was disclosed as being significant because agonist stimulation of the .alpha..sub.2 receptors was said to inhibit secretion of epinephrine and norepinephrine, while antagonism of the .alpha..sub.2 receptor was said to increase secretion of these hormones. Thus, the use of non-selective alpha-adrenergic blockers, such as phenoxybenzamine and phentolamine, is limited by their .alpha..sub.2 adrenergic receptor mediated induction of increased plasma catecholamine concentration and the attendant physiological sequelae (increased heart rate and smooth muscle contraction).
For a general background on the .alpha.-adrenergic receptors, the reader's attention is directed to Robert R. Ruffolo, Jr., .alpha.-Adrenoreceptors: Molecular Biology, Biochemistry and Pharmacology, (Progress in Basic and Clinical Pharmacology series, Karger, 1991), wherein the basis of .alpha..sub.1 /.alpha..sub.2 subclassification, the molecular biology, signal transduction (G-protein interaction and location of the significant site for this and ligand binding activity away from the 3'-terminus of alpha adrenergic receptors), agonist structure-activity relationships, receptor functions, and therapeutic applications for compounds exhibiting .alpha.-adrenergic receptor affinity was explored.
The cloning, sequencing and expression of alpha receptor subtypes from animal tissues has led to the subclassification of the receptors into .alpha..sub.1a, (Lomasney, et at., J. Biol. Chem., 266:6365-6369 (1991), rat .alpha..sub.1a ; Bruno et al., BBRC, 179:1485-1490 (1991), human .alpha..sub.1a), .alpha..sub.1b (Cotecchia, et al., PNAS, 85;7159-7163 (1988), hamster .alpha.1.sub.b ; Libert, et al., Science, (1989), dog .alpha..sub.1b ; Ramarao, et al., J. Biol. Chem., 267:21936-21945 (1992), human .alpha..sub.1b), and most recently, in a study using bovine brain, a new .alpha..sub.1c subtype was proposed (Schwinn, et al., J. Biol. Chem., 265:8183-8189 (1990); Hirasawa et al., BBRC. 195:902-909 (1993), described the cloning, functional expression and tissue distribution of a human .alpha..sub.1c adrenergic receptor; Hoehe et al., Human Mol. Genetics 1(5):349 (8/92) noted the existence of a two-allele Pst1 restriction fragment polymorphism in the .alpha..sub.1c adrenergic receptor gene; another study suggests that there may even be an alpha-1d receptor subtype, see Perez et al., Mol. Pharm., 40:876-883, 1992). Each .alpha..sub.1 receptor subtype exhibits its own pharmacologic and tissue specificities. Schwinn and coworkers noted that the cloned bovine .alpha..sub.1c receptor exhibited pharmacological properties proposed for the .alpha..sub.1a subtype. Nonetheless, based on its non-expression in tissues where the .alpha..sub.1a subtype is expressed, and its sensitivity to chloroethylclonidine, the receptor was given a new designation.
The differences in the .alpha.-adrenergic receptor subtypes have relevance in pathophysiologic conditions. Benign prostatic hyperplasia, also known as benign prostatic hypertrophy or BPH, is an illness typically affecting men over fifty years of age, increasing in severity with increasing age. The symptoms of the condition include, but are not limited to, increased difficulty in urination and sexual dysfunction. These symptoms are induced by enlargement, or hyperplasia, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra. Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the urethra.
In benign prostatic hyperplasia, the male hormone 5.alpha.-dihydrotestosterone has been identified as the principal culprit. The continual production of 5.alpha.-dihydrotestosterone by the male testes induces incremental growth of the prostate glad throughout the life of the male. Beyond the age of about fifty years, in many men, this enlarged glad begins to obstruct the urethra with the pathologic symptoms noted above.
The elucidation of the mechanism summarized above has resulted in the recent development of effective agents to control, and in many cases reverse, the pernicious advance of BPH. In the forefront of these agents is Merck & Co., Inc.s' product PROSCAR.RTM. (finasteride). The effect of this compound is to inhibit the enzyme testosterone 5-alpha reductase, which converts testosterone into 5.alpha.-dihydrotesterone, resulting in a reduced rate of prostatic enlargement, and often reduction in prostatic mass.
The development of such agents as PROSCAR.RTM. bodes well for the long-term control of BPH. However, as may be appreciated from the lengthy development of the syndrome, its reversal also is not mediate. In the interim, those males suffering with BPH continue to suffer, and may in fact lose hope that the agents are working sufficiently rapidly.
In response to this problem, one solution is to identify pharmaceutically active compounds which complement slower-acting therapeutics by providing acute relief. Agents which induce relaxation of the urethral smooth muscle, by binding to alpha-1 adrenergic receptors, thus reducing the increased adrenergic tone due to the disease, would be good candidates for this activity. Thus, one such agent is alfuzosin, which is reported in EP 0 204597 to induce urination in cases of prostatic hyperplasia. Likewise, in WO 92/0073, the selective ability of the R(+) enantiomer of terazosin to bind to adrenergic receptors of the .alpha..sub.1 subtype was reported. In addition, in WO 92/161213, hereby incorporated by reference, combinations of 5-alpha-reductase inhibitory compounds and alpha 1-adrenergic receptor blockers (terazosin, doxazosin, prazosin, bunazosin, indoramin, alfuzosin) were disclosed. However, no information as to the .alpha..sub.1a, .alpha..sub.1b, or .alpha..sub.1c subtype specificity of these compounds was provided as this data and its relevancy to the treatment of BPH was not known. Current therapy for BPH uses existing non-selective alpha-1 antagonists such as prazosin (Minipress, Pfizer), Terazosin (Hytrin, Abbott) or doxazosin mesylate (Cardura, Pfizer). These non-selective antagonists suffer from side effects related to antagonism of the alpha-1a and alpha-1b receptors in the peripheral vasculature, e.g., orthostatic hypotension and syncope.
Typically, identification of active compounds is accomplished through use of animal tissues known to be enriched in adrenergic receptors. Thus, rat tissues have been used to screen for potential adrenergic receptor antagonists. However, because of species variability, compounds which appear active in animal tissue may not be active or sufficiently selective in humans. This results in substantial wastage of time and effort, particularly where high volume compound screening programs are employed. There is also the danger that compounds, which might be highly effective in humans, would be missed because of their absence of appreciable affinity for the heterologous animal receptors. In this regard, it has been noted that even single amino acid changes between the sequence of biologically active proteins in one species may give rise to substantial pharmacological differences. Thus, Fong et al., (J. Biol. Chem., 267:25668-25671, 1992) showed that there are 22 divergent amino acid residues between the sequence of the human neurokinin-1 receptor and the homologous rat receptor. They further showed, in studies with mutant receptors, that substitution of only two amino acid residues was both necessary and sufficient to reproduce the rat receptor's antagonist binding affinity in the human receptor. Oksenberg et al., (Nature, 360:161-163, 1992) showed that a single amino-acid difference confers major pharmacological variation between the human and the rodent 5-hydroxytryptamine receptors. Likewise, Kuhse et al., (Neuron, 5:867-873, 1990) showed that a single amino-acid exchange alters the pharmacology of the neonatal rat glycine receptor subunit. This difficulty and unpredictability has resulted in a need for a compound screen which will identify compounds that will be active in humans.
These problems were solved by cloning the human adrenergic receptor of the .alpha..sub.1c subtype (ATCC CRL 11140) and the use of a screening assay which enables identification of compounds which specifically interact with the human .alpha.1c adrenergic receptor. [PCT International Application Publication Nos. WO94/08040, published 14 Apr. 1994 and WO94/10989, published 26 May 1994] As disclosed in the instant patent disclosure, a cloned human .alpha..sub.1c adrenergic receptor and a method for identifying compounds which bind the human .alpha..sub.1c receptor has now made possible the identification of selective human .alpha..sub.1c adrenergic receptor antagonists useful for treating BPH. The instant patent disclosure discloses novel compounds which selectively bind to the human .alpha..sub.1c receptor. These compounds are further tested for binding to other human alpha 1 receptor subtypes, as well as counterscreened against other types of receptors, thus defining the specificity of the compounds of the present invention for the human .alpha..sub.1c adrenergic receptor.
Compounds of this invention are used to reduce the acute symptoms of BPH. Thus, compounds of this invention may be used alone or in conjunction with a more long-term anti-BPH therapeutics, such as testosterone 5-alpha reductase inhibitors, including PROSCAR.RTM. (finasteride). Aside from their utility as anti-BPH agents, these compounds may be used to induce highly tissue-specific, localized .alpha..sub.1c adrenergic receptor blockade whenever this is desired. Effects of this blockade include reduction of intra-ocular pressure, control of cardiac arrhythmias, and possibly a host of alpha-1c receptor mediated central nervous system events.